Oligonucleotides have been used in various biological and biochemical applications. Oligonucleotides have been used as primers and probes for the polymerase chain reaction (PCR), as antisense agents used in target validation, drug discovery and development, as ribozymes, as aptamers, and as general stimulators of the immune system. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients. More recently, Kynamro® (Mipomersen sodium injectable; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) has achieved marketing clearance (2013) from the U.S. Food and Drug Administration (FDA), and is currently a treatment of homozygous familial hypercholesterolemia (HoFH). The widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for their synthesis.
Synthesis of oligonucleotides is generally performed on solid support by the repeated coupling of phosphoramidite monomers until the predetermined length and sequence is achieved. The resulting full length oligonucleotide is then cleaved from the solid support and purified with a 5′-hydroxyl protecting group left on. The industry standard 5′-hydroxyl protecting group is the 4,4′-dimethoxytrityl (DMT) group. The phosphoramidite method is well known in the art (see for example: Beaucage and Caruthers (1981) Tetrahedron Letters 22:1859-1862; McBride and Caruthers (1983) Tetrahedron Letters 24:245-248; Sinha et al. (1984) Nucleic Acids Res. 12:4539-4557 and Beaucage and Iyer (1992) Tetrahedron 48:2223-2311, each of which is incorporated herein by reference in its entirety).
Large scale synthesis of oligomeric compounds using the phosphoramidite approach is generally performed using solid phase chemistries wherein oligomeric compounds are assembled in an iterative process on a solid support. A first monomer subunit is coupled to a free hydroxyl group attached to a solid support via a series of chemical reactions. This series of chemical reactions is repeated in an iterative manner for each additional monomer subunit until an oligomeric compound having a predetermined length and base sequence is synthesized. After the oligomeric compound has been cleaved from the solid support the DMT-on oligomeric compound is purified by reverse phase liquid chromatography. When the 5′-terminal protecting group is a 4,4′-dimethoxytrityl (DMT) group the oligomeric compound is referred to as a DMT-on oligomeric compound. The 4,4′-dimethoxytrityl (DMT) group is normally left on to simplify the purification step.
Removal of 4,4′-dimethoxytrityl (DMT) protecting groups from terminal 5′-hydroxyl groups has been reported using warm conditions with mildly acidic buffers to try to limit depurination (see Salon et al., Nucleosides, Nucleotides and Nucleic Acids, 2011, 30, 271-279).
Removal of a monomethoxytrityl (MMT) group from a 5′-amino-modified oligonucleotide is performed using aqueous acid such as 20% glacial acetic acid in water (see MMT romoval, Glen Research, http://www.glenresearch.com/GlenReports/GR24-28.html).